Leishmania vaccine

ABSTRACT

The present invention relates to the use of a live mutant  Leishmania  in the preparation of a vaccine and to vaccine formulations for use in immunizing mammals, such as dogs and/or humans. The mutant  Leishmania  comprises at least one defective cysteine proteinase gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 09/402,573 filed Oct. 25, 1999, which claimspriority to PCT/GB98/00994, filed Apr. 3, 1998, which claims priority toUnited Kingdom Application No. 9706930.6, filed Apr. 4, 1997, thedisclosures of which are incorporated herein by reference in theirentireties.

The present invention relates to a Leishmania vaccine, more particularlyan attenuated live Leishmania vaccine for use in immunising mammals,such as dogs and/or humans.

The World Health Organisation estimates that Leishmania is prevalent in88 Countries of the world with a population at risk estimated at 367million with 400,000 deaths a year. Leishmania species also infect othermammals. In many countries, leishmaniasis in dogs is an importantproblem. The close proximity of humans and dogs, and the evidence thatdogs and other mammals act as reservoirs of human infection, have meantthat there has been widespread killing of dogs as a disease controlmeasure. This is clearly unsatisfactory, but also has not made asignificant impact on the spread of human disease. Thus the developmentof a vaccine would be of immense benefit to both humans and dogs.

Leishmaniasis encompasses a large spectrum of clinical diseases, whichdepending upon the parasite species and the host immune response, canhave various outcomes. In humans these clinical syndromes include singlecutaneous lesions, which may or may not spontaneously heal, and moresevere cases associated with metastasis to other cutaneous ormucocutaneous sites, as well as visceralization of the parasites leadingto a fatal infection if not properly treated. Present understanding ofthe factors that lead to this diversity of clinical symptoms has, inlarge part, come from studies using murine models (reviewed Alexander &Russell, 1992; Liew & O'Donnell, 1993). While the vast majority of mousestrains develop healing lesions when infected subcutaneously with L.major, virtually all develop non-healing lesions full of parasites wheninfected subcutaneously with L. mexicana (Roberts et al., 1989; Robertset al., 1990). Furthermore, as L. mexicana will visceralise from primarylesions in mice under the influence of genetic controls originallyidentified from studies of L. donovani, this parasite in mice offers anexcellent model system for putative vaccine studies againstdisseminating disease in a variety of susceptible genotypes.

In areas such as Southern Europe and South America where the causativeagents of visceral leishmaniasis are L. infantum and L. chagasi,respectively, the domestic dog represents the major animal reservoir ofthe disease. The incidence of canine leishmaniasis throughout SouthernEurope can range from 10% to as much as 60% (Dye et al., 1992, 1993).Once a dog develops symptoms the outcome is invariably fatal (Bray 1982;Slappendel 1988). A major question remaining to be addressed, however,is whether vaccination can effectively induce a cellular immune responseand protection against canine leishmaniasis.

It is the general consensus of opinion that acquired protective immunityagainst murine leishmaniasis, both cutaneous and visceral, is dependenton the ability to mount an IL-12 driven CD4+ T Helper 1-type response(reviewed Bogdan et al., 1996). This lymphocyte subset produces IFN-γwhich mediates protection by up-regulating macrophage inducible nitricoxide synthase (iNOS) expression and NO production which is microbicidalfor the parasites (Liew & O'Donnell, 1993). Consequently, neutralisationof IL-12 or IFN-γ, or inhibition of NO production, results in diseaseexacerbation (reviewed Bogdan et al., 1996).

The immunological pathways leading to the development of non-healingprogressive disease are less well characterised and more contentious.Thus, although a large number of studies have indicated thatsusceptibility to L. major (reviewed Liew & O'Donnell 1993; Bogdan etal., 1996), L. mexicana (Satoskar et al., 1995) and L. amazonensis(Afonso & Scott, 1993) is related to developing a Th2 response and IL-4production with down-regulation of Th1-associated activities, furtherstudies on these species, as well as L. donovani, suggest that theinability to mount a Th1 response rather than the presence of Th2response may determine susceptibility (Kaye et al., 1991; Satoskar &Alexander, 1995; Noben-Trauth et al., 1996). Nevertheless, recentstudies using IL-4 gene-deficient mice from a number of geneticbackgrounds have demonstrated a requirement for IL-4 in L. mexicanadisease progression. In the absence of this cytokine, lesions did notdevelop at the site of subcutaneous inoculation (Satoskar et al., 1995).

Proteinases have been shown to play an important role in thepathogenicity of parasitic protozoa (see Robertson et al., 1996; Coombs& Mottram, 1997). L. mexicana contains multiple, highly active cysteineproteinases (CPs), many of which are stage-regulated. The presentinventors have characterised biochemically a large number of CPs, manyof which are stage-specific (reviewed Coombs & Mottram, 1997), and haveisolated three L. mexicana CP genes; cpa, a single copy gene encoding anon-abundant cathepsin L-like CP (Mottram et al., 1992); cpb, amulticopy gene which encodes the major cathepsin L-like CPs ofamastigotes (Souza et al., 1992); and cpc, a single copy gene encoding acathepsin B-like CP (Bart et al., 1995).

In Leishmania, genes can be deleted by homologous recombination usinggene-specific targeting DNA linked to an antibiotic resistance gene,such as hyg or neo, providing positive selection. cpa null mutants havebeen generated, but have no detectable phenotype (Souza et al., 1994).cpb null mutants, however, were found to have a virulence phenotype(Mottram et al., 1996).

All life-cycle stages of the cpb null mutant can be cultured in vitro,demonstrating that the gene is not essential for growth ordifferentiation of the parasite under these conditions. The null mutant,however, exhibits a marked phenotype affecting virulence—its infectivityto macrophages is reduced 5-10 fold. Data suggest that the mutants canonly survive in a subpopulation of macrophages, but the parasites thatsuccessfully infect these macrophages grow normally. cpa/cpb double nullmutants were also created using four antibiotic selectable markers, hyg,ble, sat and pur (Mottram et al., 1996). These had a similar phenotypeto the cpb null mutant in terms of macrophage infectivity, showing thatcpa does not compensate for the loss of cpb functions in this phenotypictest.

It was also observed that subcutaneous lesions in BALB/c mice resultingfrom inoculation of the cpb null mutant appeared considerably later thanthose due to the wild-type parasites, but nevertheless lesions wereobserved (Mottram et al, 1996).

The development of a live Leishmania vaccine line by gene replacementhas previously been studied. A conditional auxotroph of L. major inwhich the dihydrofolate reductase-thymidylate synthase (dhfr-ts) genehad been deleted, was evaluated for its usefulness as a potentialvaccine. The dhfr-ts mutant was found to be an effective vaccine linefor immunizing against cutaneous Leishmania and was shown to beincapable of establishing a persistent infection or causing disease inthe most susceptible strains of mice tested. However, mild infectionswere observed after subsequent parasite challenge, which is generallyundesirable for a vaccine (Titus et al., 1995).

It is among the objects of the present invention to provide an improvedLeishmania vaccine. More particularly it is a preferred object of thepresent invention to provide a live attenuated Leishmania vaccine whichdoes not substantially display any disease manifestation afterinoculation and/or after subsequent parasite challenge.

In one aspect the present invention provides the use of a mutantLeishmania in the preparation of a vaccine, wherein the mutantLeishmania comprises at least one defective cysteine proteinase genetype, such that the mutant Leishmania is substantially incapable ofexpressing a functionally active form of said at least one cysteineproteinase.

Preferably the mutant Leishmania comprises two or more defectivecysteine proteinases and thus is substantially incapable of expressingfunctionally active forms of said two or more cysteine proteinases.

A further aspect of the invention relates to the vaccine itself.

The mutant Leishmania is preferably one which is substantially incapableof causing any disease manifestation, such as lesion formation, to amammal, and/or there is no disease manifestation in a mammal which hasbeen inoculated with the mutant Leishmania and subsequently challengedwith a further disease causing Leishmania. However, the mutantLeishmania must at least be able to survive in a mammalian host for asufficient length of time, so that an immune response may be elicitedthereto. While the present description refers mainly to the use ofpromastigotes in the preparation of a vaccine, it is to be understoodthat pure amastigotes or amastigotes in mammalian cells may be used asalternatives.

The mutant Leishmania may be selected from all species of Leishmaniaincluding L. braziliensis, L. peruviana, L. guyanensis, L. mexicana, L.major, L. amazonensis, L. infantum, L. chagasi and L. donovani,Preferably the mutant Leishmania displays cross-protection to otherLeishmania species. Thus, for example, a mammal immunised with a mutantL. mexicana as described herein may not only provide protection toinfection from disease-causing L. mexicana but also to otherdisease-causing species, as listed above.

A “defective cysteine proteinase gene” is one which is substantiallyincapable of encoding for a native cysteine proteinase or a functionalequivalent thereof. In line with common terminology cysteine proteinaseis understood to relate to and include the proteolytic enzymescontaining a nucleophilic cysteine as a member of the catalyticmachinery. This is described in detail in Barrett and Rawlings 1996.Thus, a “defective cysteine proteinase gene” means that the cysteineproteinase gene has been modified by a deletion, insertion, substitution(or other change in the DNA sequence such as rearrangement) such thatthe cysteine proteinase gene is generally incapable of expressing afunctionally competent cysteine proteinase from said gene. It will beappreciated that modification may also extend to the promoter and/ortermination region of the gene, providing that the result is that afunctionally competent cysteine proteinase from the particular gene isnot expressed.

The “defective cysteine proteinase gene may” however be capable ofexpressing a defective cysteine proteinase which is inactiveenzymically. Such a defective cysteine proteinase may however beantigenlc or immunogenic, such that a host may elicit an immune responseto the defective cysteine proteinase.

If the mutant is for example a L. mexicana mutant then said at least onecysteine proteinase gene may be selected from, for example, cpa, asingle copy gene encoding a non-abundant cathepsin L-like CP; cpb, amulticopy gene which encodes the major cathepsin L-like CPS ofamastigotes; and cpc, a single copy gene encoding a cathepsin B-like CP.

The present inventors have now also identified corresponding genes tocpa and cpb in L. infantum and their corresponding proteins (see FIGS.8-11), such that L. infantum cysteine proteinase mutants may also beproduced as described herein and used in the preparation of a vaccine.

Thus, in a further aspect the present invention provides a vaccineformulation comprising a mutant L. infantum wherein at least onecysteine proteinase gene has been made defective as described herein.Preferably the mutant L. infantum comprises two or more defectivecysteine proteinases.

CP genes of the present invention which are subsequently incapable ofexpressing a functionally competent cysteine proteinase may be rendereddysfunctional by any one or more ways for example:

-   (i) A deletion of the entire cysteine proteinase coding region of    the cp gene from a wild type Leishmania genome. The deletion should    be such so as not to substantially affect the expression of other    gene products from the leishmania parasite genome.-   (ii) A deletion of a portion of the cysteine proteinase coding    region from a wild type Leishmania genome. A “portion of the    cysteine proteinase coding region” means a polynucleotide fragment    which by its deletion from the CP coding region is sufficient to    render any CP or fragment or fragments thereof encoded and/or    expressible thereby, substantially incapable of a physiological    activity attributable to that of a functional CP produced by a wild    type parasite. The deleted portion of cp may compromise a deletion    of a small number of nucleotides, for example, 1, 2 or more    nucleotides. Such deletions within the cp gene can be achieved using    recombinant DNA technology. Thus, the translational open reading    frame (ORF) for a cp can be altered resulting in the production of a    protein which lacks the physiological functionality or functional    competence of a CP derived from wild type Leishmania. The skilled    addressee will also appreciate that such deletions in the    translational ORF of the cp gene may also give rise to a    dysfunctional gene which is substantially incapable of coding for a    functionally competent CP, truncated CP or polypeptide fragment    thereof. Such proteins/polypeptides, if produced, generally lack the    functional competence typical of the CP enzyme.-   (iii) The deletion of the or a portion of the cop gene as described    in (i) or (ii) above will leave a “gap” in the cp gene. A suitable    polynucleotide such as a gene or gene fragment thereof may be    inserted into the “gap”. Gene insertions can include genes which    express polypeptides capable of augmenting an immune response, such    as mammalian cytokines, for example, γ interferon or other genes    such as marker genes. Suitable marker genes may include but are not    restricted to genes encoding enzymes, for example thymidine kinase,    or genes encoding antibiotic resistance to such as, puromycin,    tunicamycin, hygromycin, neomycin, phleomycin, nourseothricin and    the like. Generally these genes, if any, may be employed in a cp    deletion. Mutants of the invention should be such so as not to cause    substantial deleterious or long lasting side-effects to a recipient    animal.

It is preferred however that antibiotic resistance genes are not presentin cp-mutant cell lines to be used clinically for vaccination. Thus, itis preferred to utilise a system which generates drug resistancemarker-free mutants. Such a system may involve sequential rounds oftargeted gene disruption using positive and negative selection. In thegeneration of CP double or multiple null mutants, typically, each ofsaid at least two cp genes will be targeted for disruption independentlyand subsequently multiple mutants will be generated. The hygromycin(hyg) gene may be used as a positive selectable marker for theantibiotic hygromycin. The viral HSV thymidine kinase gene (tk) may beused as a negative selectable marker in conjunction with the drugganciclovir (Lebowitz, 1994).

For example, wild type Leishmania may be transfected with a constructcontaining both the hyg and tk genes arranged in tandem in order todelete one allele of a cp gene. Selection with hygromycin will allowtransformants to be selected in which the particular cp gene has beendeleted as, for example, described previously (Mottram et al 1996). Asecond round of transfection would then be performed with a “nulltargeting fragment”, containing cp flanking DNA, that will delete thehyg/tk DNA that has been integrated into the cp locus. Cells in whichthe tk gene remains may then be killed by the ganciclovir drug, whereasmutants in which the cp gene and the drug markers have been deleted willgrow. In this manner one allele of the cp gene will have been deletedand no exogenous DNA will remain in the cp locus. The procedure willthen be repeated for the second cp allele as Leishmania is diploid, toproduce a cp-null mutant. The entire procedure may then be repeated ifnecessary in order to produce a viable cp double null mutant andrepeated again to produce viable cp triple null mutants etc.

In a preferment there is provided a Leishmania cp double or triple nullmutant comprising deletions in said cp regions within the Leishmaniagenome. The deletion should be such that coding sequences for other geneproducts of the Leishmania, upstream and/or downstream from the cpdomains, are not substantially affected. That is to say that other geneproducts ordinarily having an immunogenic function and which areexpressed in Leishmania substantially retain their immunogenic function.

The deletion generally has to be made in said cp genes in positions suchthat any mutant Leishmania within a host cell retains a sufficientimmunogenic function to elicit at least a cellular immune response (suchas cytotoxic T-cell response more preferably a Th1 cell response) in ahost animal, such as a dog or human. If the prophylactic and/ortherapeutic effect of an appropriate Leishmania mutant of the presentinvention is to be augmented, an appropriate adjuvant protein orpolypeptide, such as a cytokine, for example, γ interferon (γ IFN) canalso be employed as a component of a vaccine or pharmaceuticalcomposition of the invention.

Optionally, such cp-null Leishmania mutants may be further modified toexpress a dysfunctional form of a cysteine proteinase, such as thecysteine proteinase(s) which is/are not expressed by the mutant. Thus,the mutant may express a cysteine proteinase which is enzymicallyinactive, but which is antigenic or immunogenic. For example, to producean inactive cysteine proteinase the active site cysteine of the cp genecan be changed by site-directed mutagenesis to a glycine residue. Thismutation results in the production of a full length cysteine proteinaseenzyme that is functionally inactive. The gene encoding the inactivecysteine proteinase can then be re-introduced into a cp-null mutant byhomologous recombination using unique sequence that flank the cp gene.Similar dysfunctional cysteine proteinases can be produced by furthermutagenesis as described for example in Example 5.

In a further embodiment of the invention there is provided a host cellcomprising a cp-null Leishmania mutant of the present invention. Thehost cell may for example be a macrophage or similar cell type known tothose skilled in art.

cp-null Leishmania mutants of the present invention may be applieddirectly to the cells of an animal in vivo, or by in vitro infection ofcells taken from the said animal, which cells are then introduced backinto the animal. Leishmania cp-null mutants may be delivered to varioustissues of the animal body including muscle, skin or blood cellsthereof. The Leishmania cp-null mutant may be injected into for example,muscle or skin using a suitable syringe.

cp-null leishmania mutants for injection may be prepared in unit dosageform in ampoules, or in multidose containers. The parasites may bepresent in such forms as suspensions, solutions, or emulsions in oily orpreferably aqueous vehicles. For any parenteral use, particularly if theformulation is to be administered intravenously, the total concentrationof solutes should be controlled to make the preparation isotonic,hypotonic, or weakly hypertonic. Nonionic materials, such as sugars, arepreferred. Any of these forms may further comprise suitable formulatoryagents, such as starch or sugar, glycerol or saline. The compositionsper unit dosage, whether liquid or solid, may contain from 0.1% to 99%of parasite material.

In a further embodiment of the invention there is provided a vaccineagainst Leishmania comprising a cp-deficient Leishmania mutant. Thevaccine of the invention may optionally include a further compoundhaving an immunogenic function such as a cytokine, for example, γinterferon; or a disfunctional cysteine proteinase. The disfunctionalcysteine proteinase may for example be obtained from the cpa, cpb andcpc genes of L. mexicana as well as the corresponding cpa and cpb genesof L. infantum as disclosed herein (see FIGS. 8 and 10).

The present invention therefore also relates to the use of L. infantumcpa and cpb genes (see FIGS. 8 and 10) and to the corresponding proteinsexpressed therefrom (see FIGS. 9 and 11).

The dysfunctional cysteine proteinase(s) may be provided in the vaccineas a purified or semi-purified protein or expressed by other carrierssuch as bacteria, viruses, protozoa, as well as the particular mutant(s)Leishmania of the present invention.

In a preferred presentation, the vaccine can also comprise an adjuvant.Adjuvants in general comprise substances that boost the immune responseof the host in a non-specific manner. A number of different adjuvantsare known in the art. Examples of adjuvants may include Freund'sComplete adjuvant, Freund's Incomplete adjuvant, liposomes, and niosomesas described in WO 90/11092, mineral and non-mineral oil-basedwater-in-oil emulsion adjuvants, cytokines, short immunostimulatorypolynucleotide sequences for example in plasmid DNA containing CpGdinucleotides such as those described by Sato Y. et al. (1996); Kreig A.M. (1996).

In addition, the vaccine may compromise one or more, suitablesurface-active compounds or emulsifiers, e.g. Span or Tween.

In a further aspect of the invention there is provided the use of acp-deficient Leishmania mutant as described herein for the manufactureof a vaccine for the prophylaxis and/or treatment of Leishmaniasis. Mostpreferably, the use is in dogs or humans.

In a further aspect of the invention there is provided a method oftreating animals which comprises administering thereto a vaccinecomposition comprising a cp-deficient Leishmania mutant as describedherein to animals in need thereof. Preferably, the animals are dogs orhumans. Naturally, the vaccine formulation may be formulated foradministration by oral dosage, by parental injection or otherwise.

The invention also provides a process for preparing a Leishmaniavaccine, which process comprises admixing a cp-deficient Leishmaniamutant as herein described with a suitable carrier or adjuvant.

The mode of administration of the vaccine of the invention may be by anysuitable route which delivers an immunoprotective amount of the parasiteof the invention to the subject. However, the vaccine is preferablyadministered parenterally via the intramuscular or deep subcutaneousroutes. Other modes of administration may also be employed, wheredesired, such as oral administration or via other parental routes, i.e.,intradermally, intranasally, or intravenously.

Generally, the vaccine will usually be presented as a pharmaceuticalformulation including a carrier or excipient, for example an injectablecarrier such as saline or pyrogenic water. The formulation may beprepared by conventional means. It will be understood, however, that thespecific dose level for any particular recipient animal will depend upona variety of factors including age, general health, and sex; the time ofadministration; the route of administration; synergistic effects withany other drugs being administered; and the degree of protection beingsought. Of course, the administration can be repeated at suitableintervals if necessary.

Embodiments of the invention will now be illustrated by way of thefollowing Figures and Examples; wherein

FIG. 1 shows cutaneous lesion growth in BALB/c mice infected with 5×10⁶stationary phase L. mexicana promastigotes of wild type (•), Δcpa (∘),or Δcpb (Δ). The data are means from each group of mice; (the symbol Δis used herein to refer to a particular mutant which is defective in thenamed gene(s));

FIG. 2 shows analysis of plasma IgG1 and IgG2a levels in BALB/c mice 6months after infection with wild type (WT), Δcpa, Δcpb, or Δcpa/cpbstationary phase L. mexicana promastigotes. Values represent mean endpoint dilutions±SEM;

FIG. 3 shows Con A (5 μg/ml) or L. mexicana lysate (5 μg/ml) inducedsplenocyte proliferation responses in L. mexicana wild type(WT)-infected or Δcpb-, Δcpa/cpb-infected BALB/c mice 6 monthspost-infection. Data represented as mean stimulation index±SEM;

FIG. 4 shows IFN-γ production by cultured splenocytes removed fromBALB/c mice 9 months post-infection with wild type (WT), Δcpa, Δcpb, orΔcpa/cpb stationary phase L. mexicana promastigotes. Cytokine analysiswas performed on antigen (5 μg/ml)-stimulated (a) or Con A-stimulated(b) cultures. Non-stimulated cultures (0 μg/ml) were used as controls.Bars represent SEM;

FIG. 5 a shows IL-2 and FIG. 5 b IL-4 production by cultured splenocytesremoved from BALB/c mice 6 months post-infection with wild type (WT) orΔcpa/cpb stationary phase promastigotes. Cytokine analysis was performedon antigen (5 Δg/ml)-stimulated or non-stimulated (0 μg/ml) cultures.Bars represent SEM. FIG. 5 c shows IL-4 production by splenocytes frommice 9 months post-infection with wild type (WT) or Δcpb (N53) orΔcpb/cpa (DN) or Δcpa (H2B1) stationary phase promastigotes;

FIG. 6 a shows IL-2, FIG. 6 b IL-4 and FIG. 6 c IFN-γ production bycultured splenocytes removed from C57BL/6 mice 6 months post infectionwith wild type (WT) or Δcpa/cpb stationary phase promastigotes. FIG. 6 dshows IFN-γ production by cultured splenocytes removed from vaccinatedCBA/Ca mice (Δcpa/cpb) or naive mice (WT) 8 weeks post-challenge withwild-type parasites. Cytokine analysis was performed on antigen (5μg/ml) stimulated-or non-stimulated (0 μg/ml) cultures. IFN-γ productionby Con A (5 μg/ml)-stimulated cultures are also included (FIG. 6 c).Bars represent SEM;

FIG. 7 shows lesion growth as measured by diameter in BALB/c mice, (a,b)or C57BL/6 mice (c) non-vaccinated (∘), or vaccinated with Δcpa/cpbstationary phase promastigotes (•) 2 months (FIG. 7 a) or 4 months (FIG.7 b) before infection with wild type parasites. Bars represent SEM.Lesion growth was significantly slower until week 12 in the two monthvaccinated group (<0.05) and until week 8 and at week 12 in the 4 monthvaccinated group (<0.05). Three of the non-vaccinated mice in the latterexperiment had to be sacrificed at 10 weeks post-infection and 3 at 12weeks post-infection because of the excessive lesion growth.Consequently the lesion size data shown for the non-vaccinated mice from10 weeks onwards is artificially low as they do not take into accountthe lesions of the sacrificed mice. With C57BL/6 (FIG. 7 c), lesiongrowth-was significantly less throughout in vaccinated mice (•) comparedwith naive mice (∘).

FIG. 8 shows the DNA sequence of the Leishmania infantum cpa gene (Seq.ID No. 1);

FIG. 9 shows the predicted protein sequence of L. infantum cpa (Seq. IDNo. 2);

FIG. 10 shows nucleic acid sequence of the L. infantum cpb gene (Seq. IDNo. 3); and

FIG. 11 shows predicted protein sequence of the L. infantum cpb gene(Seq. ID No. 4).

MATERIALS AND METHODS

Parasites. Promastiaotes of L. mexicana (MNYC/BZ/62/M379) were grown inHOMEM medium, pH 7.5, containing 10% (v/v) heat inactivated foetal calfserum (HIFCS) at 25° C. as described in Mottram et al, 1992. Thefollowing antibiotics were added in combination, as appropriate, formaintenance of drug selectable markers in the cp-deficient mutants:phleomycin (Cayla, France) at 10 μg/ml, nourseothricin hydrosulphate(Hans-Knoll Institute, Thuringen, Germany) at 25 μg/ml puromycin (Sigma)at 10 μg/ml and hygromycin (Boehringer) at 50 μg/ml.

Mice. BALB/c, C57BL/6, CBA/Ca, 129Sv/Ev, C57BL/6 recombinant activatinggene-deficient mice (RAG2−/−), were bred and maintained at theUniversities of Glasgow and Strathclyde. Unless stated otherwise, groupsof up to 20 female, 8-10 week old mice were infected subcutaneously inthe shaven rump with 5×10⁶ stationary phase promastigotes of wild typeor cp-deficient L. mexicana. Groups of normally 5 but no less than 4mice were used from each group for each sample point for eachexperiment. Lesion size was measured using a slide gauge micrometer. Inthe initial experiments undertaken at the University of Glasgow lesionsvolume was measured. Thereafter at the University of Strathclyde lesiondiameters were measured and whole parasite burdens from exciseddisrupted lesions using a Neubauer haemocytometer were assessed.

Detection of Leishmania-specific antibodies by ELISA. Peripheral bloodwas obtained from infected animals by tail bleeding into heparinisedcapillary tubes. All plasma samples were stored at −20° C. prior toanalysis for specific antibody content. Leishmania-specific IgG1 andIgG2a end-point titres were measured by ELISA as previously described inSatoskar et al, 1995. Briefly, each well of an Immulon-1 microtitreplate (Dunatech Laboratories Ltd, Billingshurst, UK) was coated with 1μg of leishmanial lysate antigen (freeze/thawed wild type promastigotesin phosphate buffered saline (PBS), pH 0.9) by overnight incubation at4° C. Following incubation of serial dilutions of plasma samples for 1 hat 37° C., bound antibodies were detected by incubation with either ratanti-mouse IgG1 horseradish peroxidase conjugate or rat anti-mouse IgG2ahorseradish peroxidase conjugate (Southern Biotechnology Associates,Birmingham, Ala., USA). Binding of conjugate was visualized withtetramethylbenzidine (0.06 mg/ml) in 0.1 M sodium acetate buffer, pH5.5, containing 0.03% H₂O₂. The colour reaction was stopped by adding10% (v/v) sulphuric acid and the absorbance measured at 450 nm. Resultsare expressed as end point dilutions where the end point is defined asthe final plasma concentration which yielded an absorbance higher than anegative control plasma sample included in the assay. Comparisonsbetween groups were made with a Mann Whitney U test.

Splenocyte responses. Spleens were aseptically removed at appropriatetimes post-infection, as detailed for individual experiments, and cellsuspensions prepared by gently teasing apart the tissue in RPMI 1640supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin, 0.05 mM β-mercaptoethanol and 10% (v/v) HIFCS (Gibco,Paisley, UK). Following centrifugation at 200×g for 10 min at 4° C.,cells were resuspended in 3 ml, Boyle's solution (0.17 M Tris-HCl, pH7.2, 0.16 M ammonium chloride) at 37° C. for 3 min to deplete red bloodcells. Spleen cell suspensions were then centrifuged at 200×g for 10 minat 4° C., resuspended, washed, and resuspended in 2 ml of complete RPMI1640 medium (as above). Viable cells were enumerated by trypan blueexclusion and the suspensions adjusted to 5×10⁶ cells/ml. 100 μlaliquots of the cell suspension were added to 96-well, flat-bottomed,tissue culture plates (Costar, Cambridge, Mass.) and 100 μl aliquots ofconcanavalin A (Con A, 5 μg/ml) or L. mexicana lysate antigen (5, 10 or25 μg protein/ml) added as appropriate. Cultures were then incubated in5% CO²/95% air for 60 h at 37° C., whereupon cultures were pulsed with0.25 μCi of [³H]TdR (sp. act. 2 Ci/nmol) and incubated for a further 12h. Supernatants were collected from parallel cultures at this time forquantification of cytokine production (see below). Pulsed cells werethen harvested onto filter paper using a cell harvester (Skatron, Lier,Norway) and TdR uptake determined by liquid scintillation on a betacounter (Pharmacia LKB Biotech, Milton Keynes, UK).

IFN-γ, IL-2 and IL-4 assays. IFN-γ, IL-2 and IL-4 production bystimulated (by Leishmania antigen or Con A) and non-stimulated cellsfrom mice infected with wild type or CP-deficient parasites weremeasured by capture ELISA. Briefly, the wells of Immunol-1 microtitreplates (Dynatech Laboratories Ltd, Billinghurst, UK) were coated withcapture antibody at 2.0 μg/ml (IFN-γ R4-6A2; IL-2 JES6-1A 1.2Pharmingen, San Diego, Calif., USA; IL-4 llBll, Genzyme, Cambridge, UK)in PBS (pH 9.0) or carbonate buffer (0.05 M, pH 9.5) by overnightincubation at 4° C. Wells were then washed three times with PBS, pH7.4/0.05% Tween-20 and blocked by incubation with 10% (v/v) FCS for 1 hat 37° C. The culture supernatants and appropriate recombinant standards(rIFN-γ, rIL-2, Pharmingen; rIL-4, Genzyme) were then added toindividual wells. For standard curves, rIFN-γ (rIFN-γ(0-10 ng/ml), IL-2(0-1350 pg/ml) and rIL-4 (0-1000 pg/ml) were used. Following incubationsat 37° C. for 2 h, the wells were washed three times with PBS, pH7.4/0.05% Tween-20 and then biotinylated rat anti-mouse IFN-γ (XMG1.2,Pharmingen; used at 1 μg/ml), biotinylated rat anti-mouse IL-2(JES6-5H4, Pharmingen, used at 1 μg/ml) or goat polyclonal anti-IL-4(Genzyme; used at 1 μg/ml) were added and incubated for 1 h at 37° C.For the detection of bound biotinylated rat antibody, 100 μl ofstreptavidin-alkaline phosphatase conjugate (diluted 1/1000, Pharmingen)was added to each well for 45 min at 3720 C. and, following furtherwashing, binding was visualized with substrate consisting ofp-nitrophenyl phosphate (1 mg/ml; Sigma, UK) in glycine buffer (0.1 M,pH 10.4). The absorbance was subsequently measured at 405 nm on aTitertek Multiscan Plate Reader. For detection of bound biotinylatedgoat antibody, 100 μl of streptavidin-horseradish peroxidase conjugate(diluted 1/500, Genzyme) was added to each well for 30 min at 37° C. andfollowing further washing was incubated with tetramethylbenzidine asdescribed above. Cytokine concentrations in the cell cultures weredetermined from the standard curve (regression coefficient, r=0.990 orbetter) All assays were carried out in triplicate. Comparisons betweengroups were made using the Student's t test. P values of <0.05 wereconsidered significant.

Vaccine studies. Three mouse strains (BALB/c, CBA/Ca and C57BL/6) thatdevelop non-healing lesions when infected with L. mexicana and have beenused previously for vaccine studies were examined. The protocols foreach mouse strain were modified to reflect their response toCP-deficient mutants. Two and 4 months post-inoculation sub-cutaneouslyin the flank with 5×10⁶ Δcpa/cpb stationary phase promastigotes, BALB/cmice were infected sub-cutaneously into the shaven rump with 5×10⁶ wildtype parasites and disease progression was compared with non-vaccinatedmice. CBA/Ca and C57BL/6 mice were vaccinated with Δcpa/cpb(sub-cutaneous inoculation of 10⁷ stationary phase promastigotes) andchallenged 6 weeks later with 2×10⁵ wild type L. mexicana. Lesion growthwas monitored and compared with non-vaccinated control animals.

Example 1

Generation of L. mexicana Cysteine Proteinase Single and Double NullMutants.

L. mexicana cysteine proteinase single (Δcpa or Δcpb) and double nullmutants (Δcpa/cpb) which contain defective cpa and/or cpb genes, wereprepared according to the procedure described in Mottram et al 1996.

Example 2

Infectivity of Wild Type, Single Null and Double Null L. MexicanaCysteine Proteinase Deficient Mutants

After sub-intaneous inoculation with 5×10⁶ promastigotes suspended inphosphate buffered saline, BALB/c mice were observed for lesionformation (see FIG. 1 a).

It was observed that both the wild-type and single null mutants resultedin the generation of lesions, although notably the single null mutantproduces lesions which grow at significantly slower rates than wild-typeparasites and are about 100-fold smaller. The lesions resulting frominfection with Δcpb were slow to appear (first appearance at week 31)and very small (mean lesion volume at week 37 was 3.5 mm³). No lesionswere observed in animals which were inoculated with the cpa/cpb doublenull mutant.

Example3

Immune Response to L. mexicana Cysteine Proteinase Single and DoubleNull Mutants

Antibody response generated following infection with wild type orCP-deficient L. mexicana. Plasma levels of Leishmania-specific IgG1 andIgG2a were determined 6 months post-infection (FIG. 2). Animals infectedwith wild type L. mexicana had pronounced Leishmania-specific antibodylevels, primarily of the IgG1 subclass. Mice infected with Δcpa also hadlarge antibody titres, primarily of the IgG1 subclass but withsignificantly higher IgG2a titres than those following infection withwild type L. mexicana (p<0.01). Mice infected with Δcpb hadsignificantly less IgG1 antibody than animals infected with wild type L.mexicana (p<0.01), and there was a distinct and significant increase inthe IgG2a/IgG1 ratio. Mice infected with Δcpa/cpb had very littledetectable Leishmania-specific antibody.

In vitro splenocyte proliferative responses following infection withwild type or CP-deficient L. mexicana. Both wild type and CP-deficientmutant infected mice were able to mount antigen-specific and ConA-induced proliferative responses at all time points examined (2, 4, 6and 9 months) in all experiments. From 2 months onwards theantigen-specific and from 6 months onwards the Con A-inducedproliferative responses were significantly greater in Δcpa/cpb (p<0.05for Con A and antigen) and Δcpb (p<0.05 for Con A and antigen)inoculated mice than those mice inoculated with wild type parasites(FIG. 3). IFN-γ production by stimulated splenocytes from infected mice.IFN-γ production from splenocytes isolated from BALB/c mice 9 monthspost-infection and stimulated with parasite antigen (5 μg protein/ml) orCon A are shown in FIGS. 4 a and b. Similar results were found fordifferent amounts of antigen used and at earlier time post-infection(not shown) Antigen stimulation resulted in significantly increasedIFN-γ production in comparison with background levels (p<0.05) by allsplenocyte cultures (FIG. 4 a). However, production was significantlygreater by antigen-stimulated splenocytes from animals infected withCP-deficient mutants than with wild type L. mexicana (Δcpa and Δcpb,p<0.05; Δcpa/cpb, p<0.005). Moreover, antigen-induced IFN-γ productionwas significantly greater from splenocytes isolated from mice infectedwith Δcpb (p<0.05) and Δcpa/cpb (p<0.01) than from splenocytes isolatedfrom Δcpa-infected mice. Con A stimulation increased IFN-γ production tosignificantly greater levels than background in all splenocyte cultures.Under these conditions, splenocytes derived from mice infected with Δcpa(p<0.05) and Δcpb (p<0.02) but not Δcpa/cpb produced significantly moreIFN-γ than splenocytes derived from mice with wild type infections (FIG.4 b).

IL-2 and IL-4 production by stimulated splenocytes from infected mice.Subsequent studies concentrated on comparing the developing immuneresponse in mice infected with Δcpa/cpb and wild type parasites. Inaddition to confirming differences in splenocyte IFN-γ production, IL-2,IL-4, IL-5, IL-10 and IL-12 production was measured. No differences insplenocyte IL-5, IL-10 and IL-12 production were observed betweenΔcpa/cpb and wild type parasite infected mice, however profounddifferences in IL-2 and IL-4 production were observed at 2, and inparticular, 4 and 6 months post-infection (FIGS. 5 a and b). Whilesplenocytes from animals infected with wild type parasites failed toproduce a significant antigen-induced increase in IL-2 production, thoseinfected with Δcpa/cpb produced, following stimulation with antigen,IL-2 significantly over background (p<0.01). Antigen-stimulatedsplenocyte IL-4 production (at 6 months post infection) was significantover background for both Δcpa/cpb (p<0.001) and wild type infected(p<0.001) animals. However, the increase in splenocyte IL-4 productionwas significantly greater in wild type infected animals than Δcpa/cpbinfections (p<0.001).

Infectivity of CP-deficient mutants for other mouse strains. Δcpa/cpbparasites failed to induce lesions growth not only in C57BL/6, CBA/Caand 129Sv/Ev mice but also in RAG2-deficient C57BL/6 mice up to 6 monthspost infection (results not shown). Small lesions were, however, inducedin C57BL/6 and RAG2−/− mice by inoculation with Δcpb (results notshown). All animals infected with wild type parasites developed largenon-healing lesions. The immunological responses generated by wild typeparasites or CP-deficient mutants in the wild type mouse strains wereexamined and were similar to those observed in BALB/c mice. In C57BL/6mice, antigen-induced splenocyte cytokine production in animalsinoculated 6 months previously with Δcpa/cpb consisted entirely of IFN-γ(p<0.01) and IL-2 (p<0.05) with virtually no IL-4 produced abovebackground levels (FIGS. 6 a, b and c). However, antigen-stimulatedsplenocytes from animals inoculated with wild type parasites producednot only significant levels of IFN-γ (p<0.05) but also IL-4 (p<0.01)with little or no IL-2 above background levels. Although IFN-γ levelsfollowing antigen stimulation were similar in both groups, the increasein splenocyte IFN-γ production in antigen-stimulated Δcpa/cpb-inoculatedanimals (5.79±1.53 pg/ml) over background (and 0.11±0.05 pg/ml) was10-fold greater than that produced over background by splenocytes fromwild type-infected mice (6.03±3.25 and 0.99±0.43 pg/ml, respectively).Con A-induced splenocyte IFN-γ production was also significantly greater(p<0.05) in mice inoculated with Δcpa/cpb than in mice inoculated withwild type parasites.

Vaccine potential of CP-deficient mutants. BALB/c mice vaccinated withthe mutant parasites 2 or 4 months before infection with wild typeparasites produced slower growing lesions (FIGS. 7 a and b) whichcontained significantly fewer parasites than similarly infectednon-vaccinated mice. At week 8 post-infection with wild type L.mexicana, the parasite burdens in mice vaccinated 2 months and 4 monthspreviously with Δcpa/cpb were significantly less than non-vaccinatedmice (vaccinated, two months 2.7×10⁶±5.4×10⁵ and 4 months1.6×10⁶±3.2×10⁵; non-vaccinated, 6.2×10⁷±2.27×10⁷ and 8.4×10⁷±2.4×10⁷),(p<0.002 and p<0.001 respectively). Whereas, all non-vaccinated and thevast majority of vaccinated mice went on to develop non-healing lesions,two of the 10 mice infected 4 months after vaccination failed to developlesions up to 12 weeks post-challenge.

CBA/Ca mice were also used in a vaccine study as they have been shown tobe more amenable to vaccination than BALB/c mice and also fail todevelop lesions following challenge with Δcpb All non-vaccinated micedeveloped non-healing cutaneous lesions following infection with wildtype L. mexicana promastigotes. However, only 1 of 4 mice vaccinatedwith Δcpb and only 1 of 5 mice vaccinated with Δcpa/cpb had developedlesions 5 months after challenge (Table 1).

TABLE 1 Incidence of cutaneous lesion development in non vaccinated orCP null mutant vaccinated CBA/Ca mice infected with 2 × 10⁵ L. mexicanawild type promastigotes. No. Mice with/ without lesions Vaccine (week20) Group 1 — 5/0 Group 2 N53* 1/3 Group 3 DN* 1/4 *Mice were vaccinatedsubcutaneously with 10⁷ stationary phase promastigotes of Δcpb (N53)single null mutant or Δcpa/cpb (DN) double null mutants 6 weeks prior tochallenge infection.Efficacy of the cpb/cpa Double Null Mutant as a Vaccine

-   1. Using a similar protocol as for BALB/c mice, protection in C57BL6    mice was very significant (see FIG. 7 c). Key: □, unvaccinated mice;    •, vaccinated mice.-   2. Using a similar protocol as for BALB/c mice, protection in CBA/Ca    mice at 13 weeks post-challenge was complete. There were no lesions    in any of the 10 vaccinated mice, whereas with the unvaccinated mice    lesions first appeared 6-8 weeks post-challenge in 8 of the group of    10.-   3. Splenocytes removed 8 weeks post-challenge from CBA/Ca mice    vaccinated with the cpb/cpa double null mutant produced    significantly more IFN-gamma than did splenocytes from unvaccinated    mice (see FIG. 6 d). This showed that challenge with wild type    parasites did not cause reversion of vaccinated animals to a Th2    response.

Example 4

Expression of Active and Inactive Forms of Recombinant CPB

Details described below are for the generation, expression andpurification of active CPB. However, the same protocol may essentiallybe followed for the production of inactive CPB.

Cloning cpbg2.8 into pQE-30

PCR Amplification of Leishmania Mexicana cpb2.8

A PCR product was amplified from the lmcpb2.8 gene (Mottram et al.,1996, Proc. Natl. Acad. Sci. USA, EMBL data base number Z49962) usingprimers JH9601 and JH9602:

Primer JH9601 GGATCCGCCTGCGCACCTGCGCGCGCGA Primer JH9602AAGCTTCTACCGCACATGCGCGGACACGG

PCR Conditions: 94° C.,  4 mins x1 94° C., 30 secs 55° C., 30 secs x1572° C.,  2 mins 72° C.,  7 mins x120 μl reactions using 0.5 μl (1 unit) of VENT Polymerase (New EnglandBiolabs) with 11.1×PCR mix. After the PCR reaction was complete, Aoverhangs were added by incubating with Taq Polymerase at 72° C. for 10minutes before DNA was phenol/chloroform extracted and ethanolprecipitated.Cloning of cpbg2.8

The cpbg2.8 PCR product was cloned into the pTAG vector (R&D Systems).It was then excised from pTAG with BamHI and HindIII and cloned into thepQE-30 vector (Qiagen), using the same restriction enzymes, to giveplasmid clone pGL180. pGL180 was transformed into M15[pREP4] Esherichiacoli for expression studies.

Expression of Recombinant CPB2.8

A single colony of the E. coli M15pREP4 expression strain transformedwith the pQE-30 construct was inoculated into 8 ml of LB brothsupplemented with ampicillin (100 μg.ml⁻¹) and kanamycin (25 μg.ml⁻¹)and grown at 37° C. This culture was used to inoculate 400 ml ofLB/Anp/Kan broth and the culture grown until an OD₆₀₀ of 0.7 wasobtained. The expression of CPB2.8 was induced by the addition of IPTGto a final concentration of 1 mM. After 3 hours, the bacterial cellswere pelleted by centrifugation at 4000 g for 10 min.

Isolation of Active, Recombinant CPB2.8 from the Soluble Fraction

The bacterial pellet was resuspended in 10 ml of 50 mM Tris/HCl buffer,pH 8 containing 5 mM EDTA and 5%(w/v) sucrose. The suspension wassubjected to two rounds of freeze-thaw and six 30 second bursts on asonicator at 4° C. The bacterial lysate was centrifuged at 6000 g for 10min to pellet insoluble material and leave a supernatant fractioncontaining recombinant enzyme. This soluble fraction was dialysed for 3hours against 400 volumes of 0.1 M Tris/HCl buffer, pH 8, 0.5 M KCl, 1mM β-mercaptoethanol (buffer A). The sample was filtered through a 0.22μm filter and loaded directly at 0.2 ml.min⁻¹ onto a Ni-agarose (Qiagen)column pre-equilibrated in buffer A. The chromatography was effectedwith a stepped gradient of 0-1 mM imidazole in 0.1 M Tris/HCl buffer, pH8, 0.5 M KCl, 1 mM β-mercaptoethanol at a flow rate of 1 ml.min⁻¹.Contaminating proteins eluted from the column in 10 ml of buffer A. Therecombinant CPB2.8 enzyme was eluted with 10-20 mM imidazole in bufferA. Purity of the enzyme was assessed using silver stained SDS-PAGE. Theyield from 200 ml of culture was approximately 1-2 mg of CPB2.8 enzyme.

Isolation of Active, Recombinant CPB2.8 from the Inclusion Bodies

Following bacterial cell lysis as described above, the pelletedinclusion bodies were washed (that is, resuspended and then subsequentlypelleted again via 6000 g for 10 min) once in 10 ml of 50 mM Tris/HClbuffer, pH 8 containing 5 mM EDTA, 0.1% Triton X100, twice in 10 ml of50 mM Tris/HCl buffer, pH 8 containing 5 mM EDTA, 2 M urea, and thenfinally once in 10 ml of distilled, deionised water. The washed pelletwas solubilised at 37° C. with vigorous shaking in 10 ml of 0.1 MTris/HCl buffer, pH 8 containing 8 M urea, 10 mM DTT. The solubilisedCPB2.8 was diluted to a final protein concentration of 0.01-0.5 mg.ml⁻¹in 0.1 M Tris/HCl buffer, pH 8 containing 5 mM EDTA, 8 M urea. TheCPB2.8 enzyme was then allowed to refold to native conformation by theremoval of the chaotroph by dialysis for 15 hours against 100 volume of0.1M Tris/HCl buffer, pH 7 containing 5 mM EDTA, 5 mM cysteine. Thereducing agent was then removed by dialysis for 2 hours against 100volume of the same buffer minus cysteine. The active enzyme resultingfrom this procedure was then purified by ion exchange chromatography.

Purification of Active, Recombinant CPB2.8

The refolded CPB2.8 was filtered through a 0.22 μm filter and loadedimmediately at 1 ml.min⁻¹ onto a 1 ml Mono Q column pre-equilibrated in20 mM Tris/HCl, pH 7, 0.01% Triton X100 (buffer B). The chromatographywas developed at a flow rate of 1 ml.min⁻¹ with a stepped gradient of0-1 M NaCl in buffer B. Pro-enzyme eluted over 200-600 mM NaCl andmature enzyme with 400-600 mM NaCl. Samples were dialysed against 20 mMTris/HCl, pH 7, 0.01% Triton X-100 to remove salt, and frozen until use.Purity was assessed using silver stained SDS-PAGE. Yield of active, pureCPB2.8 was 4-5 mg from 200 ml of culture.

Example 5

Production of Recombinant Proteins of Mutated cpb and Expression ofMutated cpb in Null Mutant Parasites as Vaccine Candidates

The genes encoding different copies of CPB were mutated in a number ofways so that variants of the protein could be generated as candidatevaccines both within the attenuated leishmania themselves and asrecombinant proteins.

Constructs used for Transfections Resulting in Episomal Expression ofCPB Isoenzymes

The pX episomal shuttle vector (LeBowitz et al., 1990) was utilised fortransfection of either the L. mexicana Δcpb null mutant or Δcpa/cpbdouble null mutant. All genes were ligated to the XmaI site of pXfollowing the creation of blunt ends as required The cDNA (Souza et al.,1992) and cpb2.8 (Mottram et al., 1996) were subcloned from pBluescriptas a 2.2-kb XbaI-XhoI fragment and a 2.0-kb EcoRV fragment,respectively. Mutations were incorporated into the pBluescriptconstructs of these genes using the QuikChange Site-Directed mutagenesiskit (Stratagene) and verified by sequence analysis prior to subcloninginto pX as above. Primers used were as follows (only the sense strandprimer is shown and the mutated sites are underlined):

a) CPB2.8 mut ASM, GGTGCGTGCGGGTCGGCTGGGCGTTCTCGG b) CPB2.8 mut glycos,GCCCGAGTGCTCGAGCAGCAGTGAGCTCG c) cDNA mut 18,GACGCCGGTGAAGAATCAGGGTGCGTG d) cDNA mut 84,CGAACGGGCACCTGTACACGGAGGACAGC e) cDNAmutx3,GCTGCGATGACATGAACGATGGTTGCGACGGCGGGCTGATGCMutant

-   (a) Residue 25 of cpbg2.8 from a cysteine to a glycine. This    mutation removes the active site cysteine.-   (b) Residue 103 of cpbg2.8 from an asparagine to a serine. This    mutation removes the glycosylation site.-   (c) Residue 19 of cpb cDNA from an aspartic acid to an asparagine,    alters activity of cpb cDNA.-   (d) Residue 84 of cpb cDNA from a histidine to a tyrosine. Alters    activity of cpb cDNA.-   (e) Residues 60, 61 and 64 of cpb cDNA from aspartic acid (60),    asparagine (61) and serine (64) to asparagine, aspartic acid and    aspartic acid respectively. Alters activity of cpb cDNA.    All sequencing was performed with an ABI 373 DNA sequencer    (Perkin-Elmer) and analysed using the Wisconsin GCG package    Constructs Used for Transfections Resulting in Expression of cpb    Isoenzymes from Genes Integrated into the Genome

As an alternative to episomal expression, cpb genes (both wild type andmutated) were integrated into the L. mexicana cysteine proteinase nullmutants by homologous recombination, utilizing a construct containingthe unique 5′ and 3′ sequences flanking the cpb array (Mottram et al.,1996).

Transfection of L. mexicana

Transfection of L. mexicana promastigotes was as described previously(Souza et al., 1994; Mottram et al., 1996). Briefly, pX-based constructswere prepared using Qiagen Tip100 columns as described by themanufacturer. Transfection utilised 10 μg of DNA and 4×10⁷ late-logphase promastigotes. Following electroporation, cells were allowed torecover in 10 ml HOMEM for 24 h at 25 C and then transfectants wereselected for by transfer of cells into HOMFM containing 25 μg/ml G418.

The lines expressing the mutated cpbs and the mutated, inactive CPBsthemselves were used to vaccinate animals.

Example 6

Isolation of Leishmania Infantum cpa and cpb Genes

In Mediterranean countries, L. infantum is responsible for both visceraland cutaneous leishmaniasis with the dog serving as the principalreservoir. In Spain, for example, there is a prevalence of 0.3 VisceralLeishmaniasis cases per 100,000 inhabitants and it is calculated thatbetween 3% and 5% of all Spanish dogs are seropositive.

The L. infantum cpa and cpb genes were isolated by screening a L.infantum genomic library (Soto et al., 1993) with L. mexicana cpa(Mottram et al., 1992) and cpb (Souza et al., 1992)-specific geneprobes. The genomic library was prepared in the EMBL3 lambda vector fromtotal DNA partially digested with Sau3A1 from a L. infantum strain froma case of human Visceral Leishmaniasis (reference strain,MHOM/FR/78/LEM-75). 3 lambda clones were isolated for L. infantum cpaand 4 lambda clones were isolated for L. infantum cpb.

The complete open reading frame of the L. infantum cpa gene, togetherwith 5′ and 3′ flanking sequence, was subcloned on a 3.2 kb PstIfragment into pBluescript vector for sequencing (FIG. 8). The L.infantum cpa gene has 89% nucleotide sequence identity with the L.mexicana cpa gene (Mottram et al., 1992) and 92% identity with the L.chagasi cpa gene (Omara-Opyene and Gedamu, 1997). The predicted L.infantum protein (FIG. 9) has 86% amino acid sequence identity with L.mexicana CPA and 89% identity with the L. chagasi CPA (Omara-Opyene andGedamu, 1997). Outwith the open reading frame of the cpa genes, there issequence variation in the 5′ and 3′ flanks that will allow the design ofspecific gene targeting fragments for deletion of the L. infantum cpagene.

A PCR approach was used to amplify the complete ORF of a cpb gene fromone of the L. infantum lambda clones containing cpb. Sense and antisenseprimers were designed based on the L. mexicana and L. chagasi cysteineproteinase sequences. The sequences of the two primers for cpb were:

sense primer 5′ GTGCGAGCTGTGGCCTCTGCGT 3′ and (CPBM1) antisense primer5′ GGCGCGCGCGCACCCAAGG 3′. (CPBM2),

PCR reactions were carried out using DNA from lambda as template, 5%DMSO and Taq and KlenTaq LA polymerase (Sigma). Conditions used in PCRwere 94° 5′, 15 cycles (94° 1′, 45° 2′, 72° 2′) and final extension 72°5′. The temperature for the extension was 68° C. when the PCR usedKlenTaq LA polymerase. 1.7 kb cpb PCR products were cloned into thepGEM-T vector. Sequence from the L. infantum cpb PCR productsshowed >85% identity with the L. mexicana cpb gene (FIG. 10).

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1. A vaccine formulation comprising a mutant Leishmania infantum, saidmutant Leishmania infantum comprising at least one defective cysteineproteinase gene, such that the mutant Leishmania infantum issubstantially incapable of expressing a functionally active form of saidat least one cysteine proteinase.
 2. A vaccine formulation according toclaim 1 wherein the mutant Leishmania infantum comprises two or moredefective cysteine proteinases.
 3. A vaccine formulation according toclaim 1 wherein the mutant L. infantum comprises at least one defectiveproteinase gene selected from the group consisting of cpa and cpb.
 4. Avaccine formulation according to claim 3 wherein the at least one L.infantum defective proteinase gene is selected from the group consistingof cpa that has the sequence shown in FIG. 8 and cpb that has thesequence as shown in FIG.
 10. 5. A vaccine formulation according toclaim 1 wherein the at least one defective cysteine proteinase gene hasbeen modified by a deletion, insertion, substitution or rearrangementsuch that said at least one cysteine proteinase is substantiallyincapable of expressing a functionally competent cysteine proteinase. 6.A vaccine formulation according to claim 5 wherein said cysteineproteinase gene has been modified by deletion of all or a portion ofsaid cysteine proteinase gene.
 7. A vaccine formulation according toclaim 6 wherein a gene or gene fragment capable of expressing apolypeptide selected from the group consisting of polypeptides whichaugment an immune response and marker polypeptides is inserted into agap generated by deletion of all or the portion of said cysteineproteinase gene.
 8. A vaccine formulation according to claim 7 whereinthe polypeptide is a cytokine.
 9. A vaccine formulation according toclaim 7 wherein at least one copy of said cysteine proteinase gene hasbeen modified such that a substantially inactive form of a cysteineproteinase polypeptide is expressed.
 10. A vaccine formulation accordingto claim 1 wherein the mutant Leishmania is a drug resistant marker-freemutant.
 11. A vaccine formulation according to claim 1 that elicits acellular immune response when administered to a subject.
 12. A vaccineformulaLion according to claim 11 wherein the cellular immune responseis a Th1 cell response.
 13. A vaccine formulation according to claim 1further comprising an adjuvant and/or cytokine.
 14. A vaccineformulation according to claim 1 further comprising at least onedisfunctional cysteine proteinase, wherein said disfunctional cysteineproteinase is substantially enzymatically inactive, but which isantigenic or immunogenic.
 15. A method of vaccinating a subject againstLeishmania said method comprising administering to the subject aneffective, non-toxic amount of a vaccine formulation according toclaim
 1. 16. A method according to claim 15 wherein the method comprisesparenteral administration.